Structure And Function Of Phagocyte Proteins

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Neutrophils and other circulating phagocytes generate high levels of reactive oxygen species (ROS) in response to a variety of infectious or inflammatory stimuli, in a process known as the respiratory burst. This response is attributed to the activity of NADPH oxidase, which produces superoxide, a precursor of ROS that act as microbicidal agents and mediators of inflammation. Patients with chronic granulomatous disease (CGD) have NADPH oxidase deficiencies and suffer from enhanced susceptibility to microbial infections and aberrant inflammatory responses. This project explores cellular mechanisms regulating the respiratory burst in phagocytes and is characterizing oxidative responses of related enzymes expressed in non-immune cells. In work aimed at defining signal transduction pathways that trigger activation of the phagocyte oxidase (phox), we are using gene transfection approaches to explore roles of phospholipase D, ADP-ribosylation factor-6 (ARF-6), and its nucleotide exchange factors. Information on signaling intermediates affecting the respiratory burst may provide therapeutic strategies (pharmacological targets) designed to inhibit or enhance oxidative responses of phagocytes. In other studies, we are characterizing sources of reactive oxygen species (phox homologs) in non-myeloid tissues, notably colon, kidney, thyroid and salivary glands, mucosal surfaces, brain, and vascular tissue. In these sites, the oxidants may serve in host defense and inflammatory reactions or provide redox "second messengers" that affect gene expression patterns (proliferation responses to growth factors, differentiation, cellular senescence, apoptosis or programmed cell death, oxygen sensing). In studies on the colon-specific oxidase, we have examined expression patterns of Nox1 in colon epithelial cells and have demonstrated that Nox1 is induced by terminal differentiation or by interferon-gamma. Transduction with a retrovirus encoding Nox1 functionally replaces gp91phox, restoring stimulus-dependent superoxide release in cells co-expressing the cytosolic factors, p47phox and p67phox. Furthermore, we identified unique, colon-specific homologs of these cytosolic phox co-factors, suggesting that Nox1 is a regulated, phox-like complex acting in host defense and inflammation in the colon epithelium. In studies aimed at exploring the functional role of the renal oxidase (renox or Nox4), we have developed transgenic mouse lines for renal-specific Nox4 over-expression or suppression (antisense), under the control of dietary doxycycline. We have also engineered a targeting vector for disruption of the Nox4 gene, which is ready for cloning into embryonic stem cells. We have documented functional expression of thyroid dual oxidases (duox1 and duox2) in epithelial cells of salivary glands, airways (trachea, bronchium), and rectum, suggesting that these enzymes serve as a source of hydrogen peroxide supporting the anti-microbial activity of lactoperoxidase on mucosal surfaces. Finally, we demonstrated that cultured airway epithelial cells produce hydrogen peroxide in response to calcium signals in a duox1-dependent (antisense-inhibited) manner; this system is being developed to confirm the role of this oxidase in airway host defense.